Immunotherapy of transitional cell carcinoma of urinary bladder

Karcinom prijelaznog epitela mokraćnog mjehura najčešći je karcinom mokraćnog sustava. Klinički razlikujemo dva oblika ove bolesti: mišićno-neinvazivni i mišićno-invazivni oblik. Temeljna klinička odrednica mišićno-neinvazivnih tumora su recidivne novotvorinske promjene; 60 – 90 % mišićno-neinvazivnih tumora recidivirat će ako se liječe samo transuretralnom resekcijom (TUR). Upravo stoga se nakon TUR-a u pacijenata u kojih postoji visoki rizik od ponovne pojave ili progresije bolesti provodi intravezikalna imunoterapija BCGom (bacillus Calmette-Guerin). BCG predstavlja živi atenuirani soj Mycobacterium bovis. U primjeni BCG-a intravezikalne terapije razlikujemo indukcijsku terapiju i terapiju održavanja. Intravezikalna aplikacija BCG-a uzrokuje masivan ulazak upalnih stanica u stijenku mokraćnog mjehura te produkciju citokina detektibilnih u sluznici mjehura i u urinu, što dovodi do imunog odgovora protiv tumora. Činjenica je kako BCG uzrokuje dugotrajan i dugodjelujući imuni odgovor. Do eradikacije tumorskih stanica dolazi zbog celularnih fokusa u stijenci mjehura, a kao direktan antitumorski efektorski mehanizam navodi se direktna antitumorska aktivnost IFN (interferon) i citotoksičnost NK (engl. natural killer) stanica.Bladder cancer is the most common cancer in urinary system. There are two clinical aspects of this disease: muscle non-invasive and muscle invasive disease. Basic characteristic of non-muscle invasive disease is tumor recurrence; 60-90 % of tumors will recurr if treated by transurethral resection (TUR) only. That is the reason why patients in whom exists the risk of recurrence or progression undergo intravesical bacillus Calmette-Guerin (BCG) immunotherapy. BCG represents live attenuated Mycobacterium bovis. There are two phases in BCG therapy: induction and maintenance therapy. Intravesical BCG application causes massive agregation of immune cells in bladder wall and producton of cytokins which causes cytotoxic tumor response. Direct effector mechanism is achieved by IFN (interferon) and NK (natural killer) cell cytotoxicity

The <i>syp22-1</i> mutant plant showed abnormal floral meristem identity.

<p>(A) Wild-type (Columbia, left) and the <i>syp22-1</i> mutant (right) were grown in continuous light (CL) at 23°C for 30 days; (B) The <i>syp22-1</i> mutant is shown after 42 days. (C) <i>syp22-1</i> mutant grown under short-day conditions (SD). (D, E) Phenotypes of <i>syp22-1</i> grown under low temperature (16°C ) conditions. <i>syp22-1</i> exhibited conversion of floral meristems to inflorescence meristems under SD or low-temperature conditions (C, D, arrowheads). Aerial rosettes were also observed when grown under low-temperature condition (E, arrows).</p

Expression level of <i>FLC</i> was elevated in <i>syp22-1</i> mutants.

<p>The expression levels of <i>FLC, FT, LFY,</i> and <i>SOC1</i> in 14-day-old wild type (WT), <i>syp22-1</i>, and <i>fve-4</i> seedlings were examined by qRT-PCR. In <i>syp22-1</i> plants, expression levels of <i>FLC</i> were elevated, which resulted in downregulation of downstream flowering pathway integrators. <i>fve-4</i>, an autonomous pathway mutant, was used as a control. Results are presented as means ±S.D. (n = 3–12).</p

The <i>syp22-1</i> mutant responded normally to gibberellic acid, photoperiodic flowering induction, and vernalization treatment.

<p>(A) The number of rosette leaves in wild type (WT) and <i>syp22-1</i> mutants treated with (red) or without (blue) GA₃. Results are presented as the means ±S.D. (n = 6 plants). (B) The total leaf numbers in wild type and <i>syp22-1</i> under SD with (red) or without (blue) vernalization. Results are presented as the means ±S.D. (n = 17 plants for SD and n = 10 plants for SD+ vernalization). Flowering of <i>syp22-1</i> was delayed in SD, which was suppressed by vernalization treatment (8 weeks at 4°C).</p

Flowering Time Modulation by a Vacuolar SNARE via <em>FLOWERING LOCUS C</em> in <em>Arabidopsis thaliana</em>

<div><p>The transition of plant growth from vegetative to reproductive phases is one of the most important and dramatic events during the plant life cycle. In <em>Arabidopsis thaliana</em>, flowering promotion involves at least four genetically defined regulatory pathways, including the photoperiod-dependent, vernalization-dependent, gibberellin-dependent, and autonomous promotion pathways. Among these regulatory pathways, the vernalization-dependent and autonomous pathways are integrated by the expression of <em>FLOWERING LOCUS C</em> (<em>FLC</em>), a negative regulator of flowering; however, the upstream regulation of this locus has not been fully understood. The <em>SYP22</em> gene encodes a vacuolar SNARE protein that acts in vacuolar and endocytic trafficking pathways. Loss of <em>SYP22</em> function was reported to lead to late flowering in <em>A. thaliana</em> plants, but the mechanism has remained completely unknown. In this study, we demonstrated that the late flowering phenotype of <em>syp22</em> was due to elevated expression of <em>FLC</em> caused by impairment of the autonomous pathway. In addition, we investigated the DOC1/BIG pathway, which is also suggested to regulate vacuolar/endosomal trafficking. We found that elevated levels of <em>FLC</em> transcripts accumulated in the <em>doc1-1</em> mutant, and that <em>syp22</em> phenotypes were exaggerated with a double <em>syp22 doc1-1</em> mutation. We further demonstrated that the elevated expression of <em>FLC</em> was suppressed by <em>ara6-1</em>, a mutation in the gene encoding plant-unique Rab GTPase involved in endosomal trafficking. Our results indicated that vacuolar and/or endocytic trafficking is involved in the <em>FLC</em> regulation of flowering time in <em>A. thaliana</em>.</p> </div

The late flowering phenotype of <i>syp22-1</i> was suppressed by <i>flc-3</i>.

<p>(A) Wild type (WT), <i>syp22-1, flc-3</i>, and <i>flc-3 syp22-1</i> plants were grown in CL for 30 days at 23°C. (B) Numbers of rosette leaves are shown for wild type (WT) and mutant plants grown under the same conditions as those in (A). Results are presented as means ±S.D. (n = 6 or 7 plants). The <i>flc-3</i> mutation suppressed only the late flowering phenotype of the <i>syp22-1</i> phenotypes.</p

The <i>syp22-1</i> mutation synergistically aggravated the <i>lfy-2</i> mutation.

<p>(A) Morphology around shoot apical meristems of wild type (left, top) and mutant plants. The <i>lfy-2 syp22-1</i> double mutant (middle, bottom) exhibited a phenotype similar to that of <i>lfy-1</i> (right, top). (B) Numbers of proximal 15 lateral organs in wild type and mutants. Results are presented as the means ±S.D. (n = 12 plants). The <i>syp22-1</i> mutation alone did not markedly affect lateral organ identity, but it strongly promoted the transformation from flowers to inflorescences, when combined with the <i>lfy-2</i> mutation.</p

BEN1 and BEN2 are involved in distinct steps of early endosomal trafficking.

<p>(A,B) PIN2 immunofluorescence signals in root epidermal cells without drug treatment (A) and after ES1 treatment (36 µM, 2 h) (B). Arrowheads indicate intracellular agglomerations of PIN2 signals. (C) Quantitative analyses of intracellular accumulation of PIN2 in ES1 treated root epidermal cells. Asterisks indicate significant difference from wild type control (**: P<0.01; ***: P<0.0001 by t-test). Error bars indicate standard deviation among individual roots. N: number of cells examined. (D) Uptake of endocytic tracer FM4-64 in wild type (left) and <i>ben2</i> (right) root epidermal cells, 15 minutes after the onset of FM4-64 labeling. Magnified views of the boxed regions are indicated. Signal intensity is represented by the color code as indicated. (E) Ultrastructure of membranes associated with the Golgi apparatus in root epidermal cells of wild type and mutants. Scale bars: 20 µm in (B) for (A,B); 5 µm for (D); 300 nm for (E).</p

<i>ben</i> mutations alter the pattern of organ initiation and primordia morphology.

<p>(A,B) Localization of PIN1-GFP in developing LRPs. PIN1-GFP localizes to the anticlinal sides of young LRPs (white arrowheads) and gradually shifts its localization toward the tip of developing LRP within three days (A; yellow arrows). Relocation was less clear in the <i>ben1; ben2</i> double mutant (B). (C,D) Auxin response maxima as visualized by DR5rev::GFP reporter. Whereas sharp peaks were formed at the tips of LRPs in wild type, DR5 expression was broader in malformed LRPs of <i>ben1; ben2</i> three days after the onset of induction (C). Sharp peaks of auxin response maxima were maintained over time in wild type. In contrast, new auxin response maxima were generated in the base of LRPs in <i>ben1; ben2</i> (D). (E) A model to explain altered root architecture. PIN1 relocates towards the tip of LRP and facilitates directional auxin flow in provascular tissue (green arrows), resulting in generation of auxin maxima at the tip of LRP (green circle). PIN relocation is compromised in <i>ben1; ben2</i> double mutant. PIN1 is ectopically expressed in the epidermis by unknown mechanism, which in turn generates atypical auxin flow (blue arrows). Scale bars: 20 µm in (B) for (A,B); 50 µm in (C); 100 µm in (D).</p

Double labeling experiments reveal early endosomal localization of VPS45 in root epidermal cells.

<p>(A) Wild type VPS45-GFP (green) colocalized with FM4-64 (red) within 10 minutes after the onset of staining. Additional endocytic compartments were labeled by prolonged incubation (upper panels). Mutated VPS45-GFP carrying <i>ben2</i> mutant sequence (VPS45D129N-GFP, green) did not colocalize with FM4-64 under the same conditions (lower panels). (B) Immunostaining of BEN1 (red) and VPS45-GFP (green). Whereas wild type version of VPS45-GFP partially colocalized with BEN1 (upper left panel), <i>ben2</i> mutation abolished the colocalization (lower left panel). BEN1 and VPS45-GFP responded differently to BFA (upper right panel). Whereas BEN1 accumulates to the center of the BFA compartment in BFA-treated cells, majority of VPS45-GFP localized to the periphery of the BFA compartment. <i>ben2</i> mutation caused mislocalization of the VPS45-GFP protein (green), although it did not affect the agglomeration of BEN1 signal (lower right panel). Magnified views of the regions indicated by white squares are shown in the bottom panels. The right panels show merged images. Scale bars: 5 µm.</p